Bilirubin is a degradation product of hemoglobin. In a healthy individual, bilirubin released from aged or damaged red blood cells in the body is excreted or degraded into other derivatives. In some cases, however, an excessive amount of bilirubin occurs within the body through overproduction of bilirubin as in the case of excessive hemolysis or liver failure. Invariably, the result of an excessive amount of bilirubin within the human body is jaundice which is characterized by markedly increased levels of bilirubin in serum. There is increasing evidence that excessive amounts of bilirubin in the blood can lead to an undesirable increase in bilirubin concentration within the body cells which interferes with various cellular processes. The clinical diagnostic significance of bilirubin determination, then, in tests for liver and other related organ functions, is apparent.
In human body fluids such as bile and serum, bilirubin exists in several different forms, these forms commonly being referred to in the art as conjugated bilirubin (B.sub.c, both mono- and diconjugateed forms), unconjugated bilirubin (B.sub.u, also known as indirect bilirubin) and delta bilirubin (also known as biliprotein). The total bilirubin content B.sub.T represents the sum of all forms of bilirubin.
A variety of colorimetric assays for bilirubin are known. At one time, the most widely used assay procedure was the diazo method which employs a coupling reaction of bilirubin with a diazonium salt to form a detectable pigment. This method has its disadvantages, and many variations and improvements have been developed over the years.
Other types of assays involve the direct measurement of bilirubin which is a yellow pigment. However, this technique is susceptible to spectral interference from hemoglobin and other components of the fluid sample. It also suffers from interference from protein materials, such as albumin, to which bilirubin can bind thereby producing a shift in absorption intensity and peak wavelength.
A significant advance in the art is described and claimed in U.S. Pat. No. 4,069,017 (issued Jan. 17, 1978 to Wu et al). The assay for bilirubin described therein is carried out with a dry multilayer analytical element containing an interactive mordant in a reagent layer which binds to bilirubin thereby producing a detectable product. No other interactive composition is used in the assay. The element also comprises a porous spreading layer and a radiation-blocking layer. The interactive mordant is dispersed in a binder material, such as gelatin or its derivatives, poly(vinyl alcohol), poly(vinyl pyrrolidone) and acrylamide polymers, that is homopolymers of acrylamides (See Column 15, lines 7-39). The Examples (Column 23) in this patent teach the use of gelatin as the matrix material. Indeed, such an analytical element for the determination of bilirubin containing gelatin as the binder material has been sold for a number of years.
Another advance in the art is described in U.S. Pat. No. 4,338,095 (issued July 6, 1982 to Wu) which relates to a method for selective determination of conjugated and unconjugated bilirubin. The assay is carried out with an element similar to that described in U.S. Pat. No. 4,069,017, and contains an interactive mordant for the bilirubin dispersed in gelatin. The different forms of bilirubin are selectively detected at two or more wavelengths.
While these elements represent advances in the art, improvement is desired for the determination of bilirubin. These elements are normally sold in a cartridge containing up to 50 individual elements. It has been observed that nonuniform results are sometimes obtained among elements due to sensitivity to environmental conditions when stored for a period of time, resulting in a bias because of a change in background color.